Isolation and sequencing of a cDNA clone encoding the 85 kDa human lysosomal sialoglycoprotein (hLGP85) in human metastatic pancreas islet tumor cells.

A full length cDNA for a human lysosomal membrane sialoglycoprotein (hLGP85) was isolated as a probe of the cDNA of rat LGP85 (rLGP85) from the cDNA library prepared from total mRNA of QGP-1NL cells, a human pancreatic islet tumor cell with a high metastatic activity. The deduced amino acid sequence shows that hLGP85 consists of 478 amino acid residues (MW. 54,289). The protein has 10 putative N-glycosylation sites and 2 hydrophobic regions at the NH2- and near the COOH-termini, respectively. Thus, both domains probably constitute putative transmembrane domains. It exhibits 86% and 79% sequence similarities in amino acids and nucleic acids to rat lysosomal membrane sialoglycoprotein (rLGP85), respectively. The protein contained the short cytoplasmic tail at the COOH-terminus which does not form the glycine-tyrosine sequence (GY motif), the so-called lysosomal targetting signal.     

Effects of abdominal pressure on venous return: abdominal vascular zone conditions

The effects of changes in abdominal pressure (Pab) on inferior vena cava (IVC) venous return were analyzed using a model of the IVC circulation based on a concept of abdominal vascular zone conditions analogous to pulmonary vascular zone conditions. We hypothesized that an increase in Pab would increase IVC venous return when the IVC pressure at the level of the diaphragm (Pivc) exceeds the sum of Pab and the critical closing transmural pressure (Pc), i.e., zone 3 conditions, but reduce IVC venous return when Pivc is below the sum of Pab and Pc, i.e., zone 2 conditions. The validity of the model was tested in 12 canine experiments with an open-chest IVC bypass. An increase in Pab produced by phrenic stimulation increased the IVC venous return when Pivc-Pab was positive but decreased the IVC venous return when Pivc - Pab was negative. The value of Pivc - Pab that separated net increases from decreases in venous return was 1.00 +/- 0.72 (SE) mmHg (n = 6). An increase in Pivc did not influence the femoral venous pressure when Pivc was lower than the sum of Pab and a constant, 0.96 +/- 0.70 mmHg (n = 6), consistent with presence of a waterfall. These results agreed closely with the predictions of the model and its computer simulation. The abdominal venous compartment appears to function with changes in Pab either as a capacitor in zone 3 conditions or as a collapsible Starling resistor with little wall tone in zone 2 conditions.     

Cyclization reaction catalyzed by branching enzyme.

The action of branching enzyme (EC 2.4.l.l8) from Bacillus stearothermophilus on amylose was analyzed. The enzyme reduced the molecular size of amylose without increasing the reducing power. This result could not be explained by the normal branching reaction model. When the product was treated with glucoamylase (an exo++-type amylase), a resistant component remained. The glucoamylase-resistant component was easily digested by an endo-type alpha-amylase or by isoamylase plus glucoamylase. These results suggested that the glucoamylase-resistant component was a cyclic glucan composed of alpha-1,4- and alpha-l,6-glucosidic linkages. In other words, it was suggested that branching enzyme catalyzed cyclization of the alpha-l,4-glucan chain of the amylose molecule to form an alpha-l,6-glucosidic linkage, thereby forming two smaller molecules. Mass spectrometry also supported the cyclic nature of the product.     

Isolation of Serratia marcescens mutants which could overproduce and excrete Escherichia coli alkaline phosphatase and beta-lactamase

Serratia marcescens mutants, which excrete Escherichia coli alkaline phosphatase (APase) encoded by the plasmid-bearing phoA gene, were isolated after mutagenesis by N-methyl--nitro-N-nitrosoguanidine. These mutants produced two to four times as much APase as did the parent strain under a phosphate-limiting condition, and more than 70% of the enzyme was released into the culture medium. In addition, overproduction and excretion of beta-lactamase was observed in these mutants.     

W-heterochromatin of chicken; its unusual DNA components,late replication,and chromatin structure

About 65% of DNA in the chicken W chromosome has been shown to consist ofXhoI andEcoRI family repetitive sequences. These sequences showed remarkable delay in the electrophoretic mobility at low temperature on a polyacrylamide gel. Three dimensional structures of the 0.7-kbXhoI and the 1.2-kbEcoRI family repeating units were estimated to be irregular solenoids using a computer program based on wedge angles of all the 16 dinucleotide steps. Fluorescencein situ hybridization demonstrated that these two family sequences were localized in a major heterochromatic body in an interphase nucleus. Incorporation of bromodeoxyuridine into the W chromosome in the synchronous culture of MSB-1 cells occurred about 1 h later than the peak of S phase. The chromatin structure formed alongXhoI andEcoRI family sequences was suggested to be different from the total chromatin or chromatin containing the β-actin gene sequence in that the linker DNA lengths of the former were significantly longer. Fractionation of theHaeIII-digested MSB-1 nuclei yielded a chromatin fraction in whichXhoI family sequences were partially enriched. Several DNA-binding proteins showing higher affinity for theXhoI family sequence were present in this fraction.     

Some Electrical Properties of a Nuclear Membrane Examined with a Microelectrode

Electrical potential and resistance were measured with microelectrodes in in situ and isolated nuclei of gland cells of Drosophila flavorepleta. The nucleus-cytoplasm boundary was found to be rather impermeable to ion diffusion. It presents a resistance of the order of 1 Ω cm² and sustains a "resting" potential, the nucleoplasm being about 15 mv negative with respect to the cytoplasm. Both the resistance and potential appear to be associated with the nuclear membrane: the potential declines to zero and the resistance to a fraction of its original value, when the membrane is perforated experimentally.     

Na+-dependent glucose transporter SGLT1 is localized in the apical plasma membrane upon completion of tight junction formation in MDCK cells

SGLT1, an isoform of Na⁺-dependent glucose transporters, is localized at the apical plasma membrane in the epithelial cells of the small intestine and the kidney. In the present study we examined its location in SGLT1 cDNA-transfected MDCK cells, which form an epithelial sheet connected by tight junctions in culture. Formation of tight junctions was monitored by staining for occludin, an integral tight junction protein. In the cells demarcated by an uninterrupted occludin meshwork, SGLT1 was specifically localized at the apical plasma membrane, showing that SGLT1 has a signal to accomplish this restricted localization. In the cells with little or no occludin accumulation in the tight junction, however, SGLT1 was present along the entire aspect of the plasma membrane. Similar distribution of SGLT1 was observed in the cells as long as the occludin meshwork remained incomplete. These observations sugget that apical localization of SGLT1 occurs upon the completion of the uninterrupted meshwork of tight junctions.     

Characterization of a gene cluster for glycogen biosynthesis and a heterotetrameric ADP-glucose pyrophosphorylase from Bacillus stearothermophilus.

A chromosomal region of Bacillus stearothermophilus TRBE14 which contains genes for glycogen synthesis was cloned and sequenced. This region includes five open reading frames (glgBCDAP). It has already been demonstrated that glgB encodes branching enzyme (EC 2.4.1.18 [H. Takata et al., Appl. Environ. Microbiol. 60:3096-3104, 1994]). The putative GlgC (387 amino acids [aa]) and GlgD (343 aa) proteins are homologous to bacterial ADP-glucose pyrophosphorylase (AGP [EC 2.7.7.27]): the sequences share 42 to 70% and 20 to 30% identities with AGP, respectively. Purification of GlgC and GlgD indicated that AGP is an alpha2beta2-type heterotetrameric enzyme consisting of these two proteins. AGP did not seem to be an allosteric enzyme, although the activities of most bacterial AGPs are known to be allosterically controlled. GlgC protein had AGP activity without GlgD protein, but its activity was lower than that of the heterotetrameric enzyme. The GlgA (485 aa) and GlgP (798 aa) proteins were shown to be glycogen synthase (EC 2.4.1.21) and glycogen phosphorylase (EC 2.4.1.1), respectively. We constructed plasmids harboring these five genes (glgBCDAP) and assayed glycogen production by a strain carrying each of the derivative plasmids on which the genes were mutated one by one. Glycogen metabolism in B. stearothermophilus is discussed on the basis of these results.     

The Purification of a GroEL-Like Stress Protein from Aerobically Adapted Campylobacter jejuni

From plate cultures of Campylobacter jejuni grown in room air a particulate protein of 62 kDa was isolated by ion-exchange chromatography. The protein had a square shape from the side view but when viewed from the top it had a star-shaped structure. The molecular size of the whole particle determined by gel filtration was 850 kDa which suggested the presence of 14 subunits of 62 kDa in each particle. The N-terminal 37 amino residues showed more than 80% homology with the sequence of these heat shock protein (HSP) 60 homologs of Chlamydia trachomatis, Helicobacter pylori, and Escherichia coli (GroEL). This protein is immunologically cross-reactive with the antiserum for the 60-kDa HSP of Yersinia enterocolitica. Production of the 62-kDa protein increased under heat stress and growth in an aerobic atmospheric environment. From these observations we concluded that the 62-kDa protein is a Campylobacter stress protein (Cj62) which belongs to the HSP 60 family.